4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl

Endpoint summary

Description of key information

Executive Summary on repeat dose toxicity studies with 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl

The repeat dose toxicity of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl was determined in two subacute studies (OECD 407, rat, 28d, oral administration) and in a reproductive screening assay performed in rats (OECD 421, oral) (1,2).

In the first subacute study, daily oral administration of 100, 300 or 1000 mg/kg of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl to rats for 4 weeks was clinically tolerated with no mortality, clinical signs, changes of body weight, food and water consumption, and no effect on hematology and clinical chemistry parameters in weeks 5 and 7. However, in histopathology, moderate to massive vacuolation and degeneration in the zona fasciculate of the adrenal gland at all dose levels was observed with a comparable degree. Females were more affected than males and control animals did not show any incidence. In the ovary, mild to massive cytoplasmic vacuolation of interstitial cells was detected in all rats of all treatment groups. There was no clear dose-dependency referring to the severity of this finding. No signs of reversibility were discernable after the 2-week treatment free period in the 1000 mg/kg group. Concerning the degeneration and necrosis of single cells in the adrenal cortex, the severity was slightly increased.

In a second subacute study a daily oral administration of 3, 10, and 30 mg of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl/kg to rats for 28 days was clinically tolerated with no mortality, clinical signs, changes of body weight, or relevant changes of food consumption. In histopathology, female rats showed dose-dependent vacuolation in the adrenal cortex and vacuolation of interstitial cells of the ovary were observed down to the lowest dose of 3 mg/kg bw, where still one out of five females was affected. Males were not affected and controls did not show incidences. No signs of reversibility were discernable after the 2-week treatment free period. With respect to severity, cytoplasmic vacuolation of the Zona fasciculata was more pronounced than in the main kill females at 30 mg/kg, as 2 animals exhibited a marked degree. In addition, two females showed a minimal to mild degeneration/ necrosis of inner cortical cells which was not seen in main kill animals. In the ovaries findings were comparable to main kill rats.

Cytoplasmic vacuoles in the adrenal cortex are often indicative for an early degenerative lesion and might reflect impaired steroidogenesis, resulting in excess storage of non-metabolized steroid precursors (3). A variety of lipophilic substances can cause this impaired steroidogenesis. For the ovary, it is also described that inhibition of the synthesis of steroid hormones may allow the accumulation of lipids in the ovarian stromal cell cytoplasm to give rise to histological appearances of fatty change (4). However,there exist also other factors such as spontaneous, age-related changes (especially in rats) or stress from various causes that could spontaneously attribute to the cortical vacuolization (5).

To assess possible functional impairment on fertility and development caused by the effects seen in the subacute studies, a reproductive screening assay was performed in rats (6). In this assay, 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl was administered daily at 1, 3, and 10 mg/kg bw for a treatment period up to 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females.

In males, no clinical signs were observed in any treatment group or control animals during the entire study period. In females, isolated incidences of alopecia in one control and one MD group female and slight piloerection in one MD and one HD female were observed for very few days and therefore were considered to be incidental. None of the females showed signs of abortion or premature delivery.

There were no test item treatment related effect on body weight, body weight gain, and food consumption observed in the dose groups during the study period. In males and females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group.

There were no biologically significant effects on fertility and reproductive parameters. At necropsy, few specific macroscopic changes were recorded for the male and female animals, which were not considered to be of test item treatment relevance based on microscopic examination.

Under the conditions of this study, the test item caused no histological changes indicating toxicity in adrenal glands and reproductive organs based on the pathology evaluation. In the control group, cortical vacuolation was observed in 8 of 10 males and 3 of 10 females with minimal to mild incidence. In the HD group (10 mg/kg bw), 6 of 10 males and 4 of 10 females showed minimal to mild vacuolation. Thus, vacuolation in the adrenal cortex was considered to be not test item related. There were no incidences for necrosis or degeneration in the adrenal gland noted after 63 days oral exposure.

Overall the results obtained in the reproductive screening assay clearly demonstrate that there was no functional impairment of the adrenal glands on fertility and development in rats (male/female) at 10 mg/kg bw/day after 63 days of treatment (6). Male rats did not show any effect in the subacute studies up to 30 mg/kg bw (2).

Taking all these data into account, the NO(A)EL for female rats at 10 mg/kg bw/day and the NOEL for male rats at 30 mg/kg bw are fully justified based on the finding of the mild necrosis found in two females at 30 mg/kg bw/day 2 weeks after end of treatment.

References

1.  4 week oral toxicity study in rats plus a 2 week treatment-free recovery period, 2012

2.      4 week oral toxicity study in rats plus a 2 week treatment-free recovery period, 2013

3.          Capen CC, DeLellis RA, Yarrington JT. Adrenal Cortex. In: Haschek and Rousseaux’s handbook of toxicologic pathology. Third Edition. Amsterdam; Boston: Academic Press; 2013. p. 690–2.

4.          Greaves P. Histopathology of preclinical toxicity studies: interpretation and relevance in drug safety studies. 4th ed. Amsterdam ; Boston: Elsevier/AP; 2012. 886 p.

5.          NTP. Adrenal Gland, Cortex - Vacuolization, Cytoplasmic. In: Nonneoplastic Leasion Atlas [Internet]. [cited 2017 Aug 31]. Available from: https://ntp.niehs.nih.gov/nnl/endocrine/adrenal/cxvacuol/index.htm

6.    4 week oral toxicity study in rats plus a 2 week treatment-free recovery period, 2017

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Sep 06, 2011 - Mar 13, 2012

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted October 03, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Purity: >99.90%

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species: Rat
Strain: Crl: WI (Han)
Breeder: Charles River, Germany

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 8w
- Weight at study initiation: 230 (m), 171 (f)
- Fasting period before study:
- Housing: grouped
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1°C
- Humidity (%): 37 - 65 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally by gavage, once daily, 7 times a week for 4 weeks.
The volume of administration was 5 mL/kg body weight. The volume of administration per
animal was calculated by means of the LIM-System.

Vehicle:

other: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water): lab standard vehicle
- Concentration in vehicle: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): -
- Purity: analytical grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The quantification of the test item in the dosing
formulations was performed using a HPLC method with UV
detection. During the course of the study each dosing formulation
(including control) of two preparations periods were sampled and
analyzed at the beginning and the end of usage, resulting in 4 time
points of formulation analysis.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once daily, 7 times a week for 4 weeks

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

0 mf/kg: 20 (10 m/ 10 f)
100 mg/kg: 10 (5 m/ 5 f)
300 mg/kg: 10 (5 m/ 5 f)
1000 mg/kg: 20 (10 m/ 10 f)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: recovery
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

Clinical Signs
Mortality, the behavior and appearance of each animal were checked twice daily at
working days and once at off days, preferably at the same time (s) each day. Symptoms
were recorded with the LIM-System

Motor Activity
On day 28, one hour after administration, the rats (the numerical first 5 males and 5
females per group) were removed from their home cages and their motor activity was
recorded in special motor activity cages over 60 minutes at 5 minutes intervals. The
number of movements was evaluated by counting the number of interruptions of photo
beams (7 beams on the x-axis, 4 beams on the y-axis and 7 beams on the z-axis). These
parameters are called “counts” and given in numbers. The system was also calculating the
“on-time” (minutes), “off-time” (minutes), the “total distance” the animal was moving
(meter), “rearing” (number), and “rearing time” (minutes). The data was transferred into
the LIM-System. Assignment of the rats to the individual measurements followed a
randomization schedule. On each measuring day, measurements were conducted
simultaneously in 10 rats.

FOB
A functional observational battery was performed in the numerical first 5 males and 5
females per group before the first exposure (day 0), a week thereafter (day 7), and in week
4 (day 28). On day 28, the functional observational battery was performed after the motor
activity measurement. Animal numbers and microchips were not identified for the
laboratory staff performing the functional observational battery. Therefore, the observers
did not know to which treatment group the rats belong.
The following parameters were examined in the cage, during removal of the cage, in the
observation arena and during handling: palpebral closure, ease of removal and handling
from cages, muscle tone, lacrimation, salivation, piloerection, fur appearance, mobility,
arousal, gait, approach response, touch response, click response, tail pinch response,
righting reflex, pupil response, raising, raising behavior, defecation and urination (number
of fecal boluses, feces consistency, number of urine pools, urine stain size), hind limb foot
splay, fore limb and hind limb grip strengths, internal body temperature, catalepsies,
posture, vocalization, convulsions, stereotypy, and any abnormalities.
The observed parameters were recorded in the LIM-System.

Body Weight
Each rat was weighed before treatment and thereafter once a week. The parameter was
recorded with the LIM-System.

Food consumption
Food consumption was determined once a week by weighing the food per cage which had
not been consumed. The parameter was recorded with the LIM-System.

Water consumption
Water consumption was determined twice a week by measuring the water per cage which
had not been consumed. The parameter was recorded with the LIM-System.

Laboratory tests
In weeks 5 and 7 blood was taken under inhalation anesthesia from the rats listed below.
The blood samples were divided for hematological and clinical-chemical examinations.
Before blood sampling the animals were kept in metabolism cages for the collection of
urine for approximately 18 hours without food.

HEMATOLOGY
Red blood cells (erythrocytes) /pL RBC (a)
Hemoglobin g/dL HGB (a)
Hematocrit % HCT (a)
Mean cell volume fL MCV (a)
Mean hemoglobin content pg MCH (a)
Mean hemoglobin concentration g/dL MCHC (a)
Platelets /nL PLT (a)
Reticulocytes % RET% (a)
Absolute number of reticulocytes /nL RET (a)
White blood cells (leukocytes) /nL WBC (a)
Absolute number of neutrophilic granulocytes /nL NEUT (a, b)
Absolute number of lymphocytes /nL LYM (a, b)
Absolute number of eosinophilic granulocytes /nL EOS (a, b)
Absolute number of basophilic granulocytes /nL BASO (a, b)
Absolute number of monocytes /nL MONO (a, b)
Absolute number of large unstained cells /nL LUC (a, b)
Neutrophilic granulocytes % NEUT% (a, b)
Lymphocytes % LYM% (a, b)
Eosinophilic granulocytes % EOS% (a, b)
Basophilic granulocytes % BASO% (a, b)
Monocytes % MONO% (a, b)
Large unstained cells % LUC% (a, b)
Prothrombin time sec PT sec (c)
Prothrombin time % PT % (c)
Partial thromboplastin time sec PTT (c)

a) ADVIA 120, Siemens Medical Solutions GmbH (Bad Nauheim, Germany)
b) Visual differentiation by a microscope, ZEISS (Oberkochen, Germany)
c) Coasys Plus, Roche Diagnostics (Mannheim, Germany)


Clinical chemistry
Sodium mmol/L NA ISE, indirect (a)
Potassium mmol/L K ISE, indirect (a)
Calcium mmol/L CA CPC (a)
Chloride mmol/L CL ISE, indirect (a)
Inorganic phosphate mmol/L IP Direct phosphomolybdate (a)
Glucose mmol/L GLUC Glucose-Hexokinase (a)
Urea mmol/L UREA Urease-glutamatic DH (a)
Creatinine μmol/L CREA Jaffé, without deproteinization (a)
Total bilirubin μmol/L TBIL Vanadat-Oxidation (a)
Cholesterol mmol/L CHOL CHOD-PAP (enzymatic) (a)
Triglycerides mmol/L TRIG GPO-PAP (enzymatic) (a)
Bile acids μmol/L BA Enzymatic color (a)
Total protein g/L TP Biuret (a)
Albumin g/L ALB Bromcresol green (a)
A/G ratio - A/G ratio calculated LIMS
Alanine aminotransferase U/L ALAT Kinetic UV, IFCC with P-5-P (a)
Aspartate aminotransferase U/L ASAT Kinetic UV, IFCC with P-5-P (a)
Alkaline phosphatase U/L AP Kinetic color, IFCC AMP (a)
Glutamate dehydrogenase U/L GLDH Kinetic UV, GSCC (DGKC) (a)
pH value - pH Reflectometry (b)
Protein PRO Reflectometry (b)
Glucose GLU Reflectometry (b)
Bilirubin BIL Reflectometry (b)
Blood BLO Reflectometry (b)
Urobilinogen URO Reflectometry (b)
Ketone KET Reflectometry (b)
Sediment SED Visual (c)
Specific gravity - SG Refractometry (d)

a) ADVIA 1650, Autoanalyzer, Siemens Medical Solutions Diagnostics GmbH, (Bad Nauheim, Germany)
b) Clinitek ADVANTUS, Reflection Spectrophotometer,Siemens Medical Solutions Diagnostics GmbH,
(Bad Nauheim, Germany)
c) Microscope Olympus, BX40F, Olympus Optical CO, LTD, (Hamburg, Germany)
d) Refractometer, Krüss, (Hamburg, Germany)

Gross Pathology
At the time scheduled the rats were anesthetized by a carbon dioxide air mixture and
exsanguinated by opening the abdominal vessels. They were necropsied and examined for gross
pathological alterations.
All findings were recorded with the LIM System.

Body and Organ Weights
The body and organs weights were recorded with the LIM-System. Based on the absolute organ
weights the relative organs weights (related to 100g body weight) were calculated. For all
animals the following weights were determined:
The following weights were determined:
Terminal body weight (after exsanguination)
Heart
Liver
Kidneys (together)
Spleen
Thymus
Testes (together)
Prostate
Uterus
Ovaries (together)
Adrenals (together after fixation)
Thyroids with Parathyroids (together after fixation)
Brain (after fixation)
Seminal vesicles
Epididymides (together)


Histopathology
The organs and tissues of main kill animals were fixed, histotechnically processed and examined
as listed below. The following abbreviations are used:
D Davidson’s fluid
F approx. 10 % formaldehyde solution
H.E. hematoxilin-eosin
mD modified Davidson’s fluid
 to be performed


Main kill
Adrenal (2)
Aorta
Bone with knee joint (os femoris)
Bone with bone marrow (sternum, femur)
Brain (cerebrum, cerebellum, brain stem)
Esophagus
Eye (2)
Heart F
Intestine, large
Cecum
Colon
Rectum
Intestine, small
Duodenum
Jejunum
Ileum
Kidney (2)
Larynx
Liver (left lateral and right medial lobe)
Lung (with mainstem bronchi)
Lymph nodes
mandibular (2)
mesenteric
Mammary gland (inguinal)
Micro transponder
Muscle, skeletal (thigh)
Nasal turbinates
Nerve, optic (2)
Nerve, sciatic
Pancreas
Parathyroid (2)
Peyer’s Patches
Pituitary
Reproductive organs, female
Ovary (2)
Oviduct (2)
Uterus (cornu/corpus/cervix)
Vagina
Reproductive organs, male
Epididymis (2)
Prostate
Seminal vesicle
Testis (2)
Salivary gland (2)
(submandibular, parotid, sublingual)
Skin (inguinal)
Spinal cord (cervical, thoracal, lumbal)
Spleen
Stomach (proventricular, fundic, pyloric)
Thymus
Thyroid (2)
Tongue
Trachea
Ureter (2)
Urinary bladder
Zymbal's gland (2)
All tissues showing abnormalities

For recovery animals tissue fixation was performed as for the main kill group.
The adrenals, female reproductive organs (ovary, uterus, vagina) and pituitary were investigated
in all dose groups of the main kill and in recovery animals.
All histopathology findings were recorded with the LIM-System.

Sacrifice and pathology:

The animals were necropsied, examined for gross pathological alterations, the weights of
selected organs were recorded and histotechnical procedures and histopathological examinations
were performed.

Statistics:

Body weight, body temperature, food and water consumption, organ weights, hematology,
clinical chemistry, motor activity, functional observational battery (numerical parameters):
To compare the treatment groups with the control group, the following statistical procedures
were applied separately for each sex and each measuring point. To take the number of dose
groups into account all the test procedures used maintain a multiple significance level of =
0.05.
Absolute body weight, body weight gain (differences to baseline values on day 1), body
temperature, food and water consumption, organ weights (relative and absolute), clinical
pathology parameters (hematology, clinical chemistry serum parameters, specific gravity and
urine weight), motor activity (total time period of 60 minutes), the dose groups were compared
with those of the control, using the multiple two-sided Dunnett-Test (Dunnett 1955,1964).
For all numerical parameters of the FOB, dose groups and the control groups were compared,
using the non-parametric 2-sided Kruskal-Wallis test (Hollander, 1973) followed by the
Wilcoxon-test rank sum test (Hollander, 1973).
For all parameters (except urinalysis) mean values, standard deviation, and number of animals
(N) were calculated.
For each numerical parameter of the FOB also minimum, maximum, median, and the frequencies
of scores were calculated. The descriptive parameters were listed as incidences per treatment
group in a table.
Software: Body weight, body temperature, food and water consumption, organ weights,
hematological parameters, clinico-chemical parameters (except the urinalysis parameters), motor
activity and the FOB parameters (except the frequencies of scores) were evaluated within the
LIM-System. The urinalysis examinations will be recorded with the LIM-System.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment- or dose-dependent changes of body weight, body weight gain, or food consumption were observed.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No treatment- or dose-dependent changes of body weight, body weight gain, or food consumption were observed.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Slight changes of water consumption were observed. As no clear dose-correlation was observed, the slight water consumption changes were not considered to be treatment related.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology, clinical chemistry and urinalysis did not reveal any toxicological relevant
alterations.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Hematology, clinical chemistry and urinalysis did not reveal any toxicological relevant
alterations.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The functional observational battery was performed once predose, and on days 7 and 28.
No treatment-related changes in autonomous, neuromuscular, sensomotoric, central
nervous system, or internal body temperature were observed. Measurement of motor
activity at the end of the treatment period (day 28) did not reveal any treatment related
effects.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy only spontaneous alterations were observed. There was no toxicologically relevant effect on body or organ weights.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination of the dose groups revealed cytoplasmic vacuolation of the
adrenal cortex, especially in female rats, combined with slight parenchymal degeneration
and single cell necrosis. Cytoplasmic vacuolation was also observed in interstitial cells of
the ovary. All investigated dose groups were affected in a comparable manner. The
cytoplasmic vacuolation in the adrenal and ovary probably reflects steroidogenesis
impairment. No signs of reversibility were discernable after the 2 week treatment free
period.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

not determinable

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

adrenal glands

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Daily oral administration of 100, 300 or 1000 mg/kg of to rats for 4 weeks was clinically tolerated with no mortality, clinical signs, changes of body weight,
food and water consumption, and no effect on hematology and clinical chemistry parameters in weeks 5 and 7. However, at histopathology, vacuolation and degeneration in
the adrenal gland at all dose levels was observed with a comparable degree. Females were more affected than males. No signs of reversibility were discernable after the 2-week
treatment free period in the 1000 mg/kg group. This finding is considered to be adverse as it probably reflects impaired steroidogenesis.
Therefore, a NOEL (no effect level) or NOAEL (no observed adverse effect level) could not be established at the doses of 100, 300, and 1000 mg/kg.

Executive summary:

 

Objective

4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl is an industrial chemical which needs to be registered in the European Union according to the REACH regulation . Such registrations require data on repeat dose toxicity, i.e. a subacute toxicity study in rats with oral administration.

 

Study Design

4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl was administered orally by gavage, once daily 7 times a week for 4 weeks to 3 groups of male and female Crl:WI (Han) rats at doses of 100, 300 or 1000 mg/kg body weight. A similarly constituted control group received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium).

The control and high dose groups consisted of 10 male and 10 female rats each. The low and mid dose groups consisted of 5 male and 5 female rats each. At the end of the treatment period 10 (5 male and 5 female) rats per group were scheduled for necropsy. The remaining rats of groups 1 and 4 were scheduled for a 2-week recovery period.

 

At the end of the treatment or recovery periods, all animals were subjected to a detailed necropsy and selected organ weights were recorded. Histopathological examinations were performed on selected organs of control and high dose animals (end of treatment period) and kidneys of intermediate and recovery groups.

 

Results

Formulation analysis revealed that the dose groups were sufficiently exposed to the test item: The animals received the anticipated concentrations.

 

All animals survived the treatment period and recovery until their scheduled necropsy. No treatment-related clinical signs were observed. No treatment- or dose-dependent changes

of body weight, body weight gain, or food consumption were observed.

 

Slight changes of water consumption were observed. As no clear dose-correlation was observed, the slight water consumption changes were not considered to be treatment related.

The functional observational battery was performed once predose, and on days 7 and 28. No treatment-related changes in autonomous, neuromuscular, sensomotoric, central

nervous system, or internal body temperature were observed. Measurement of motor activity at the end of the treatment period (day 28) did not reveal any treatment related

effects.

 

Hematology, clinical chemistry and urinalysis did not reveal any toxicological relevant alterations.

 

At necropsy only spontaneous alterations were observed. There was no toxicologically relevant effect on body or organ weights.

 

Histopathological examination of the dose groups revealed cytoplasmic vacuolation of the adrenal cortex, especially in female rats, combined with slight parenchymal degeneration

and single cell necrosis. Cytoplasmic vacuolation was also observed in interstitial cells of the ovary. All investigated dose groups were affected in a comparable manner. The

cytoplasmic vacuolation in the adrenal and ovary probably reflects steroidogenesis impairment. No signs of reversibility were discernable after the 2 week treatment free

period.

 

Conclusions

 

Daily oral administration of 100, 300 or 1000 mg/kg to rats for 4 weeks was clinically tolerated with no mortality, clinical signs, changes of body weight,

food and water consumption, and no effect on hematology and clinical chemistry parameters in weeks 5 and 7. However, at histopathology, vacuolation and degeneration in

the adrenal gland at all dose levels was observed with a comparable degree. Females were more affected than males. No signs of reversibility were discernable after the 2-week

treatment free period in the 1000 mg/kg group. This finding is considered to be adverse as it probably reflects impaired steroidogenesis.

Therefore, a NOEL (no effect level) or NOAEL (no observed adverse effect level) could not be established at the doses of 100, 300, and 1000 mg/kg.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 07, 2012 - Feb 20, 2013

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted October 03, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Purity: >99.90%

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species: Rat
Strain: Crl: WI (Han)
Breeder: Charles River, Germany

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany
- Age at study initiation: 8w
- Weight at study initiation: 233 (m), 165 (f)
- Fasting period before study: no
- Housing: grouped
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1°C
- Humidity (%): 37 - 65 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally by gavage, once daily, 7 times a week for 4 weeks.
The volume of administration was 5 mL/kg body weight. The volume of administration per
animal was calculated by means of the LIM-System.

Vehicle:

other: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water): lab standard vehicle
- Concentration in vehicle: 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium)
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): -
- Purity: analytical grade

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The quantification of the test item in the dosing
formulations was performed using a HPLC method with UV
detection. During the course of the study each dosing formulation
(including control) of two preparations periods were sampled and
analyzed at the beginning and the end of usage, resulting in 4 time
points of formulation analysis.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once daily, 7 times a week for 4 weeks

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

30 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

0 mf/kg: 20 (10 m/ 10 f)
3 mg/kg: 10 (5 m/ 5 f)
10 mg/kg: 10 (5 m/ 5 f)
30 mg/kg: 20 (10 m/ 10 f)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: recovery
- Post-exposure recovery period in satellite groups: 2 weeks
- Section schedule rationale (if not random): random

Positive control:

no

Observations and examinations performed and frequency:

Clinical Signs
Mortality, the behavior and appearance of each animal were checked twice daily at
working days and once at off days, preferably at the same time (s) each day. Symptoms
were recorded with the LIM-System


Body Weight
Each rat was weighed before treatment and thereafter once a week. The parameter was
recorded with the LIM-System.

Food consumption
Food consumption was determined once a week by weighing the food per cage which had
not been consumed. The parameter was recorded with the LIM-System.

Gross Pathology
At the time scheduled the rats were anesthetized by a carbon dioxide air mixture and
exsanguinated by opening the abdominal vessels. They were necropsied and examined for gross
pathological alterations.
All findings were recorded with the LIM System.

Body and Organ Weights
The body and organs weights were recorded with the LIM-System. Based on the absolute organ
weights the relative organs weights (related to 100g body weight) were calculated. For all
animals the following weights were determined:
The following weights were determined:
Terminal body weight (after exsanguination)
Heart
Liver
Kidneys (together)
Spleen
Thymus
Testes (together)
Prostate
Uterus
Ovaries (together)
Adrenals (together after fixation)
Thyroids with Parathyroids (together after fixation)
Brain (after fixation)
Seminal vesicles
Epididymides (together)


Histopathology
The organs and tissues of main kill animals were fixed, histotechnically processed and examined
as listed below.


Main kill
Adrenal (2)
Aorta
Bone with knee joint (os femoris)
Bone with bone marrow (sternum, femur)
Brain (cerebrum, cerebellum, brain stem)
Esophagus
Eye (2)
Heart F
Intestine, large
Cecum
Colon
Rectum
Intestine, small
Duodenum
Jejunum
Ileum
Kidney (2)
Larynx
Liver (left lateral and right medial lobe)
Lung (with mainstem bronchi)
Lymph nodes
mandibular (2)
mesenteric
Mammary gland (inguinal)
Micro transponder
Muscle, skeletal (thigh)
Nasal turbinates
Nerve, optic (2)
Nerve, sciatic
Pancreas
Parathyroid (2)
Peyer’s Patches
Pituitary
Reproductive organs, female
Ovary (2)
Oviduct (2)
Uterus (cornu/corpus/cervix)
Vagina
Reproductive organs, male
Epididymis (2)
Prostate
Seminal vesicle
Testis (2)
Salivary gland (2)
(submandibular, parotid, sublingual)
Skin (inguinal)
Spinal cord (cervical, thoracal, lumbal)
Spleen
Stomach (proventricular, fundic, pyloric)
Thymus
Thyroid (2)
Tongue
Trachea
Ureter (2)
Urinary bladder
Zymbal's gland (2)
All tissues showing abnormalities

For recovery animals tissue fixation was performed as for the main kill group.
The adrenals, female reproductive organs (ovary, uterus, vagina) and pituitary were investigated
in all dose groups of the main kill and in recovery animals.
All histopathology findings were recorded with the LIM-System.

Sacrifice and pathology:

The animals were necropsied, examined for gross pathological alterations, the weights of
selected organs were recorded and histotechnical procedures and histopathological examinations
were performed.

Statistics:

All parameters were analyzed separately for each sex and time. To take the number of dose
groups into account, all the test procedures used maintain a multiple significance level of
alpha= 0.05.

Absolute body weight, body weight gain (differences to baseline values on day 0), food
consumption, and organ weights - relative and absolute - of the dose groups were
compared with those of the control, using the multiple two-sided Dunnett-Test (Dunnett
1955, 1964). When the parameters were compared in the recovery period between the
control (group 1) and high dose (group 4), the standard t-test (Winer, 1970) was used.
Software: Body weight gain, food consumption and organ weights were evaluated within
the LIM-System.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs, changes of body weight, and body weight gain were
observed in any dose group of both genders during the treatment and recovery period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unchanged during the treatment period in both genders, and in
females during the recovery period. The males showed a slightly decreased food
consumption during the recovery period that is not considered biologically relevant.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy only spontaneous alterations were observed. Toxicological relevant body or organ weight deviations were not observed in any dose group of
main kill or recovery animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At histopathology examination the female rats of all groups exhibited cytoplasmatic vacuolation in the zona fasciculata of the adrenal gland. The males were not affected. There was a clear dose dependency
with regard to incidence and severity as one female at 3 mg/kg was affected to a mild degree; all females at 10 mg/kg showed a minimal to mild degree and all females at 30 mg/kg exhibited a moderate degree. The high dose recovery females showed a vacuolation of the zona fasciculata which was more pronounced than the main kill females as a moderate to massive degree was noted in all animals. Two females of the recovery group showed minimal to mild degeneration/ necrosis of inner cortical cells in addition.
In the ovary, minimal cytoplasmic vacuolation of interstitial cells was detected in one rat at 3 mg/kg, a mild degree was seen in four rats at 10 mg/kg and a moderate degree in all 5 rats at
30 mg/kg. In recovery animals at 30 mg/kg incidence and degree of cytoplasmic vacuolation of interstinal cells was consistent with the main kill animals at this dose.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

no

Lowest effective dose / conc.:

10 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

adrenal glands

Conclusions:

Daily oral administration of 3, 10, or 30 mg/kg of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl to rats for 4 weeks was clinically tolerated with no mortality, clinical signs, changes of body weight, or
relevant changes of food consumption.

However, at histopathology, alterations in the adrenal gland and ovary in female rats were observed at all dose levels. Dose-dependent vacuolation in the adrenal cortex and
vacuolation of interstitial cells of the ovary were observed, at the low dose of 3 mg/kg one rat was affected. No signs of reversibility were discernable after the 2-week treatment free
period. The cytoplasmic vacuolation in the adrenal and ovary might reflect persistent metabolic changes due to impairment of steroidogenesis and/or retention of the xenobiotic.

Therefore, for the females a NOEL (no effect level) or NOAEL (no observed adverse effect level) could not be established. For the males, a NOEL could be established at 30
mg/kg under the conditions of this study.

Executive summary:

 

Objective

4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyli s an industrial chemical which needs to be registered in the European Union according to Annex VIII of the REACH regulation (>10-100 tons per annum). Such registrations require data on repeat dose toxicity, i.e. a subacute toxicity study in rats with oral administration.

 

Study Design

4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl was administered orally by gavage, once daily, 7 times a week for 4 weeks to 3 groups of male and female Crl:WI (Han) rats at doses of 3, 10 or 30 mg/kg. A similarly constituted control group received the vehicle, 0.25% aqueous hydroxypropyl methylcellulose (Methocel® K4M Premium), and served to generate contemporary control data.

The control and high dose groups consisted of 10 male and 10 female rats each. The low dose and mid dose groups consisted of 5 male and 5 female rats each. At the end of the

treatment period, 10 (5 males and 5 females) rats were scheduled for necropsy. The remaining rats of groups 1 and 4 were scheduled for a 2-week recovery period. The rats

were gang-housed under conventional conditions.

 

All rats were subjected to macroscopic and histopathological examinations. Selected organs were weighed from each rat at the end of the treatment period.

 

Results

 

Formulation analysis revealed that the dose groups received the anticipated concentrations and no test material was detected in the control formulations.

All animals survived the treatment and recovery period.

 

No treatment-related clinical signs, changes of body weight, and body weight gain were observed in any dose group of both genders during the treatment and recovery period.

Food consumption was unchanged during the treatment period in both genders, and in females during the recovery period. The males showed a slightly decreased food

consumption during the recovery period that is not considered biologically relevant.

At necropsy only spontaneous alterations were observed. Body and organ weight determinations revealed no relevant deviations in any dose group of main kill or recovery

animals.

At histopathology the female rats of all dose groups (3, 10, and 30 mg/kg) exhibited a cytoplasmatic vacuolation in the zona fasciculata of the adrenal gland. Incidence and

severity were clearly dose-dependent as one female at 3 mg/kg was affected to a mild degree; all females at 10 mg/kg showed a minimal to mild degree and all females at

30 mg/kg exhibited a moderate degree. The males were not affected.

At the end of recovery, the high dose (30 mg/kg) females showed a moderate to massive vacuolation of the zona fasciculate. Severity was more pronounced in the main kill

females. Two females of the recovery group showed minimal to mild degeneration/necrosis of inner cortical cells in addition.

In the ovary, minimal cytoplasmic vacuolation of interstitial cells was detected in one rat at 3 mg/kg, a mild degree was seen in four rats at 10 mg/kg and a moderate degree in all 5

rats at 30 mg/kg. At the end of recovery, high dose (30 mg/kg) recovery animals had the same incidence and degree of cytoplasmic vacuolation of interstitital cells as the main kill

animals of this dose level.

 

 

Conclusions

Daily oral administration of 3, 10, or 30 mg/kg of 4-Ethyl-2',3'-difluor-4''-propyl-1,1':4',1''-terphenyl to rats for 4 weeks was clinically tolerated with no mortality, clinical signs, changes of body weight, or

relevant changes of food consumption.

 

However, at histopathology, alterations in the adrenal gland and ovary in female rats were observed at all dose levels. Dose-dependent vacuolation in the adrenal cortex and

vacuolation of interstitial cells of the ovary were observed, at the low dose of 3 mg/kg one rat was affected. No signs of reversibility were discernable after the 2-week treatment free

period. The cytoplasmic vacuolation in the adrenal and ovary might reflect persistent metabolic changes due to impairment of steroidogenesis and/or retention of the xenobiotic.

 

Therefore, for the females a NOEL (no effect level) or NOAEL (no observed adverse effect level) could not be established. For the males, a NOEL could be established at 30

mg/kg under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

excellent as it also includes reproductive screening data from an OECD 421 compliant assay

System:

urinary

Organ:

adrenal glands

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In the repeated dose toxicity studies histopathological findings were observed in the adrenal gland. These findings were considered to be not sufficient for classification and labelling